It has been estimated that up to 80% of chronic infections can be attributed to biofilm formation, a differentiation process in which bacterial cells become sessile, embed themselves in an extracellular matrix and finally form a macrocolony. Cells within biofilm communities often escape treatment with traditional antibiotics and aggravate the course of disease. Biofilm formation is a complex and highly regulated process involving a central second messenger, cyclic di-GMP (c-di-GMP), that controls many of the key events during differentiation. The underlying signaling mechanisms and pathways are largely unknown. Cyclic di-GMP and enzymes for its production and degradation are unique to eubacteria, and therefore represent attractive targets for the development of novel therapeutics against bacterial infections, a long-term goal of the proposed work. We will set out to study the structure, function and regulation of key enzymes in biofilm formation. In Specific Aims 1 and 2, we focus on distinct subfamilies of cyclases and phosphodiesterases that catalyze the synthesis and turnover of c-di-GMP, respectively. X-ray crystallography and small-angle scattering in combination with enzymatic assays will be used to decipher the activation and regulatory mechanisms of these multi-domain proteins. Insight into the conformational plasticity and regulation will be invaluable for the design and optimization of small molecule inhibitors. Specific Aim 3 focuses on uncovering signaling pathways and regulators by the identification of protein-protein interactions using proteomics and genetic screens. The results of these studies will elucidate the basic signaling reactions and networks that control biofilm formation, and will provide the basis for bicochemical and pathway-oriented small molecule screens. Infectious diseases are one of the foremost causes of mortality in developed countries. Persistent chronic infections have been associated with a phenomenon in which bacteria settle down on natural surfaces (e.g. heart valves, lungs, or ears) or medical devices (e.g. catheters, implants) and embed themselves in a so called biofilm, causing insuperable obstacles for traditional antimicrobial treatments. The process appears to be highly controlled, and by studying the enzymes and underlying regulatory principles we hope to provide novel starting points for the development of therapeutics.